KRAS-targeted Drugs R&D Service

RAS is one of the most frequently mutated oncogenes in human cancer. KRAS is the isoform most frequently mutated, which constitutes about 85% of RAS mutations. As the most frequently mutated RAS isoform, KRAS is intensively studied in the past years.

In the formulation of KRAS integrated research plan, Medicilon has in-depth communication with customers. The backbone of scientific research has combined the characteristics of each case with years of practical experience and technical accumulation, and carefully submitted high-quality experimental plans and results to customers. Medicilon provides KRAS-targeted drug discovery, CMC research (API + formulation), pharmacodynamics research, PK study, safety evaluation and other services.

Targeting the untargetable KRAS^[1]

Introduction of KRAS

• RAS is a family of GTPase proto-oncogenes, comprising three closely related RAS isoforms: HRAS, KRAS and NRAS. From all of the RAS isoforms, KRAS is most frequently mutated, followed by NRAS and then HRAS. KRAS mutations are particularly frequent in the pancreatic, lung and colorectal cancers. In cancer, the most frequently mutated residues are G12, G13, and Q61. KRAS protein exists as two splice variants, KRAS4A and KRAS4B, in which KRAS4B is the dominant form in human cells.
  KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) gene is a proto-oncogene that encodes a GTP/GDP-binding protein that belongs to the GTPase RAS family.
  The KRAS protein acts as molecular switchs that cycle between a GDP-bound inactive state and a GTP-bound active state. KRAS protein switches between an inactive to an active form via binding to GTP and GDP, respectively.
  Although the KRAS protein harbors both intrinsic nucleotide exchange and GTP hydrolysis, its cellular signaling state arises from activation by guanine exchange factors (GEFs), such as son of sevenless (SOS) and Ras guanyl nucleotide-releasing protein, which catalyze GTP loading and deactivation by GTPase activating proteins (GAPs), such as p120GAP and neurofibromin (NF1), which stimulate GTP hydrolysis.
  

  KRAS GTPase cycle^[1]

Structure of KRAS

• KRAS protein contains four domains. The first domain at the N-terminus is identical in the three RAS forms, and the second domain exhibits relatively lower sequence identity. Both regions are important for the signaling function of the KRAS protein and jointly form the G-domain. KRAS protein has a molecular weight of 21 kDa, and is made up of six beta-strands (forming the protein core) and five alpha-helices, which form two major domains: the G-domain and the C-terminal. The G domain of KRAS, comprised of residues 1-166, includes the GTP-binding pocket, a region within which is essential for the interactions between the putative downstream effectors and GTPase-activating proteins (GAPs). The G domain is highly conserved and contains switch I and switch II loops, which are responsible for GDP-GTP exchange. The C-terminal, a hypervariable region including the CAAX (C= cysteine, A = any aliphatic amino acid, X = any amino acid) motif,  guides posttranslational modifications and determines plasma membrane anchoring. This region plays an important role in the regulation of the biological activity of RAS protein.
  

  Crystal structure KRAS^[2]

  

  2D depiction of the secondary structure of KRAS^[2]

Signal Pathway of KRAS Signaling

• KRAS is one of front-line sensors that initiate the activation of an array of signaling molecules, allowing the transmission of transducing signals from the cell surface to the nucleus, and affecting a range of essential cellular processes such as cell differentiation, growth, chemotaxis and apoptosis. In addition to the aforementioned GTP/GDP binding, the activation of KRAS signaling is now known as a multi-step process that requires proper KRAS post-translation, plasma membrane-localization and interaction with effector proteins.
  The signal transduction of the KRAS protein does not exclusively occur at the plasma membrane. Activation of downstream signaling pathways by KRAS can also be triggered by signals from subcellular compartments, such as the endoplasmatic reticulum and the Golgi apparatus.
  In response to extracellular stimuli, the conversion from inactive RAS-GDP to active RAS-GTP further promotes the activation of various signaling pathways, which includes MAPK pathway, PI3K pathway and the Ral-GEFs pathway, among them the MAPK pathway is the best characterized. It is known that RAS-GTP directly binds to RAF protein, recruiting RAF kinase family from cytoplasm to membranes, where they dimerize and become active. The activated RAF subsequently carries out a chain of phosphorylation reactions to its downstream substrates, namely MEK and ERK, and propagates the growth signal.
  

  The major KRAS effector pathways^[1]

Crystallization Studies of KRAS Protein

• High Throughput Screen of Crystallization
  More than 1,000 screen conditions
  
• Shanghai Synchrotron Radiation Facility
  Medicilon is involved in the design, construction and management of Shanghai Synchrotron Radiation Facility, an industrial beamline for macromolecular crystallography. Macromolecular beamline was open on July 2009.
  

  Macromolecular Crystallography for Industrial Use

  Superior beamline and serviceLower costs3.5 GeV storage ringYear‐round operationVery close to Zhangjiang High-Tech Park and Pudong airport.
• Case study
  KRAS-G12D
  

  Screening of KRAS^G12D

  

  Synchrotron X-ray diffraction data of KRAS^G12D
  KRAS-G12D with MRTX1133
  

  Screening of the co-crystallization

  

  Synchrotron X-ray diffraction data of the co-crystallization

      KRAS-G12D

  

  Compare the Structure of KRAS^G12D with 7RPZ, the green is PDB ID 7RPZ and the pink is the data from Medicilon. The data are very correlated.

      KRAS-G12D with MRTX1133

  

  Compare the Structure of KRAS^G12D co-crystallization with MRTX1133 (7RPZ, PDB), the green strucuture is PDB ID 7RPZ and the cyan is data from Medicilon. The data are very correlated.

  KRAS-G12D with MRTX1133

  

  Structure of KRAS^G12D co-crystallized with MRTX1133 with GDP-bound

In Vitro Studies of KRAS-targeted Drugs

• In vitro functional assays are crucial for the practical evaluation of a candidate KRAS-targeted drug in the initial stages of research and development. These assays offer scientific evidence for validating KRAS-targeted drug activity, and providing preliminary evidence that supports therapeutic efficacy. As such, they play a key role in the decision-making process in KRAS-targeted drug candidate selection.
  KRAS Cellular Assay
  Medicilon have validated cytotoxicity assays for KRAS mutant cell lines, both 2D and 3D assays could be used for evaluation of KRAS inhibitors.
  

  2D cell proliferation assays detected through CellTiter-Glo

• Cell Cytotoxity Assay (3D)
  NCI-H358 (Lung, KRAS^G12C) Cell Cytotoxity Assay (3D)
  

  Cytotoxicity determined by photomicrography after 288 h treatment with drugs in NCI-H358 cells at concentrations starting from 1 µM and 1:3 serial dilution.

• NCI-H358 (Lung, KRAS^G12C) Cell Cytotoxity CTG Assay (2D; 3 days)
  

  Cytotoxicity determined by CTG after 72 h treatment with drugs in NCI-H358 cells at concentrations starting from 1 µM and 1:3 serial dilution.

• NCI-H358 (Lung, KRAS^G12C) Cell Cytotoxity CTG Assay (3D;12 days)
  

  Cytotoxicity determined by photomicrography after 288 h treatment with drugs in NCI-H358 cells at concentrations starting from 1 µM and 1:3 serial dilution.

• Protein-based Assay
  

  IC₅₀ screening of test compounds on Kras G12C-SOS1 Binding

• Guanine Nucleotide Exchange Assay
  
  KRAS was incubated in a solution containing 1 mM BODIPY FL-GDP, 20 mM HEPES pH 7.6, 10 mM EDTA, 20 mM ammonium sulfate and 1 mM DTT for 48 hours at 4°C.                 The reaction was stopped with the addition of 20 mM MgCl₂. The BODIPY-FL-GDP loaded KRAS protein is concentrated to remove BODIPY-FL-GDP. Panel A: MoA for the assay

Pharmacology Evaluation of KRAS-targeted Drugs

• KRAS Mutation - CDX Model

  Cancer Type	Cell Lines
  KRAS G12C	MIA PaCa-2, NCI-H358, UM-UC-3, Calu-1
  KRAS G12D	GP2D, SW1990, AsPC-1
  KRAS G12V	SW480, CAPAN-1, NCI-H727
  KRAS G13D	LoVo, HCT-116, HT15

  Medicilon Case: CDX - KRAS Mutation (G12C)
  
  PDX Key Mutation/Overexpression/Resistance

  GENE	PDX ID
  KRAS Mutation	PDXM-060C (p.G12V), PDXM-069C (p.G12V),PDXM-075C (p.G12D), PDXM-076C (p.G13D),PDXM-212Li (p.G12D)
  TP53 Mutation	PDXM-060C (p.R273H), PDXM-072C(p.Y234H)
  PIK3CA Mutation	PDXM-075C (p.H1047L), PDXM-092Ga (p.E545G)
  BCR-ABL Fusion	PDXM-242Le
  ERBB2 Overexpression	PDXM-069C, PDXM-016C, PDXM-060C, PDXM-087C, PDXM-104C…
  ...	...

  Resistance*	PDX ID
  Docetaxel + Cisplatin	PDXM-271O (Ovarian cancer)
  VDLP + MA + CVAD	PDXM-293Le (Leukemia)
  Radiation	PDXM-311(H&N)
  ...	...

  * note: these resistance models are not related to KRAS mutation

• Medicilon Case: PDX -- KRAS Mutation (G12D)
  
  Medicilon Case: AMG-510 Resistant Model - Calu-1 (G12C)
  

  Wild Type Lung Cancer Model

  

  Resistant Lung Cancer Model (established through in vivo treatment cycle twice (P2), two months each cycle. Show here is the P3 results.

Pharmacokinetic (PK) Studies of KRAS-targeted Drugs

• Medcilon provides high quality quantification assays for key parameters in KRAS-targeted drugs PK study, presenting accurate results.
  Medicilon Case: Pharmacokinetics of KRAS-PDEδ Inhibitors
  KRAS-PDEδ protein-protein interaction represents an appealing target for cancer therapy. A series of potent PROTAC PDEδ degraders were designed and synthesized. The most promising Compound 17f is a PROTAC PDEδ degrader for the treatment of KRAS mutant colorectal cancer. Compound 17f provided a new chemical tool or lead compound for navigating the druggablility of KRAS-PDEδ interaction. Compound 17f achieved significant tumor growth inhibition in the SW480 colorectal cancer xenograft model. This proof-of-concept study provided a new strategy to validate the druggability of KRAS-PDEδ interaction and offered an effective lead compound for the treatment of KRAS mutant cancer.
  

  PROTAC strategy and KRAS-PDEδ inhibitor Compound 17f^[3]

• Pharmacokinetic (PK) studies of Compound 17f were evaluated in Sprague-Dawley (SD) rats. After ip administration dosing at 50 mg/kg, the concentrations of Compound 17f in plasma were analyzed. These assays were conducted by Medicilon. The half-life of 17f was approximately 5.1 h and the peak concentration C_max was 564 ng/mL. Despite its relatively large size (MW = 723), Compound 17f could be effectively absorbed and achieved a sufficient plasma exposure in rats, with the area under the curve (AUC) value of 4710 h·ng/mL.
  

  PK parameters of Compound 17f in rats^[3]

Medicilon Assisted Projects

• XNW14010
  On May 2022, the State Drug Administration approved the clinical application of XNW14010, a new class 1 anti-tumor drug from Evopoint Biosciences Co., Ltd. (hereinafter referred to as "Sinovent"), which is intended for the treatment of patients with advanced solid tumors with KRAS G12C mutation.XNW14010 is a highly selective small molecule KRAS G12C protein covalent binding inhibitor independently developed by Sinovent. As Sinovent's partner, Medicilon provided comprehensive preclinical research services (including pharmacokinetics and safety evaluation) for the development of XNW14010, providing strong support for the project's clinical approval.
• References：
  [1] Pingyu Liu, et al. Targeting the untargetable KRAS in cancer therapy. Acta Pharm Sin B. 2019 Sep;9(5):871-879. doi: 10.1016/j.apsb.2019.03.002.
  [2] Tatu Pantsar. The current understanding of KRAS protein structure and dynamics. Comput Struct Biotechnol J. 2019 Dec 26:18:189-198. doi: 10.1016/j.csbj.2019.12.004.
  [3]Junfei Cheng, et al. Discovery of Novel PDEδ Degraders for the Treatment of KRAS Mutant Colorectal Cancer. J Med Chem. 2020 Jul 23;63 (14):7892-7905. doi: 10.1021/acs.jmedchem.0c00929.
  [4]Gongmin Zhu, et al. Role of oncogenic KRAS in the prognosis, diagnosis and treatment of colorectal cancer.  Mol Cancer. 2021 Nov 6;20(1):143. doi: 10.1186/s12943-021-01441-4.
  [5]Tamas Yelland, et al. Stabilization of the RAS:PDE6D Complex Is a Novel Strategy to Inhibit RAS Signaling. J Med Chem. 2022 Feb 10;65 (3):1898-1914.  doi: 10.1021/acs.jmedchem.1c01265.
  [6]Timothy H Tran, et al. KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activa-tion. Nat Commun. 2021 Feb 19;12(1):1176. doi: 10.1038/s41467-021-21422-x.